Effects of six pyrimidine analogs on the growth of Tetrahymena thermophila and their implications in pyrimidine metabolism

Tetrahymena are ciliated protists that have been used to study the effects of toxic chemicals, including anticancer drugs. In this study, we tested the inhibitory effects of six pyrimidine analogs (5-fluorouracil, floxuridine, 5’-deoxy-5-fluorouridine, 5-fluorouridine, gemcitabine, and cytarabine) on wild-type CU428 and conditional mutant NP1 Tetrahymena thermophila at room temperature and the restrictive temperature (37°C) where NP1 does not form the oral apparatus. We found that phagocytosis was not required for pyrimidine analog entry and that all tested pyrimidine analogs inhibited growth except for cytarabine. IC50 values did not significantly differ between CU428 and NP1 for the same analog at either room temperature or 37°C. To investigate the mechanism of inhibition, we used two pyrimidine bases (uracil and thymine) and three nucleosides (uridine, thymidine, and 5-methyluridine) to determine whether the inhibitory effects from the pyrimidine analogs were reversible. We found that the inhibitory effects from 5-fluorouracil could be reversed by uracil and thymine, from floxuridine could be reversed by thymidine, and from 5’-deoxy-5-fluorouridine could be reversed by uracil. None of the tested nucleobases or nucleosides could reverse the inhibitory effects of gemcitabine or 5-fluorouridine. Our results suggest that the five pyrimidine analogs act on different sites to inhibit T. thermophila growth and that nucleobases and nucleosides are metabolized differently in Tetrahymena.


Introduction
Tetrahymena thermophila are unicellular, ciliated protists whose function as a model eukaryote has contributed to the discovery of catalytic RNA, telomerase and telomere structure, and the first histone acetyltransferase, among numerous other contributions [1][2][3].In addition to their important role in the lab, Tetrahymena species living in diverse natural freshwater habitats have been used to indicate levels of pollutants present in ecosystems and to examine chemical toxicities [4,5], including classes of anticancer drugs [4][5][6][7].Among these anticancer drugs, we are particularly interested in pyrimidine analogs [6,8] as their ability to block the growth of Tetrahymena allows for the study of nucleotide uptake and metabolism in this species.Moreover, because Tetrahymena lacks de novo nucleotide synthesis pathways [9,10] we may gain further insight into the metabolism of these compounds in this organism by manipulating the exogenous supply of nucleotides [11].
Because Tetrahymena cannot synthesize their own nucleotides de novo (review in [8]), they must obtain nucleobases, nucleosides, and nucleotides from food sources or their environment.Studies have shown that uracil, uridine, deoxyuridine, cytidine, deoxycytidine, CMP, dCMP, UMP, and dUMP could each effectively serve as the lone pyrimidine source to support the growth of T. pyriformis in defined medium [9,[11][12][13].These observations suggest that most cytidine and uridine derivatives, except for cytosine which is inert in supporting the growth of T. pyriformis [9,11,12], may be deaminated and aminated to form necessary pyrimidines.Moreover, comparisons of T. pyriformis doubling time in defined medium suggested that uracil and uridine are equally effective in supporting Tetrahymena growth [11].
In contrast to the effective pyrimidine sources mentioned earlier, methylated pyrimidines, such as thymine, thymidine, TMP, and 5-methyluridine could not function as the lone pyrimidine source, suggesting that T. pyriformis lacks a pathway to demethylate these thymidine derivatives [11][12][13].
As a unicellular organism, how nutrients are obtained and enter Tetrahymena cells is also of great interest [14].In addition to the distinct phagocytosis through their oral apparatus, Tetrahymena has at least four other pathways of endocytic uptake through their peripheral membrane [15].While phagocytosis plays an important role in obtaining nutrients in Tetrahymena, there are also indications that some amino acids, nucleobases, and nucleosides may be absorbed through their peripheral membrane system or endocytosis [16][17][18].Rasmussen first reported that at a growth condition where food vacuoles formed slowly, the doubling time of T. pyriformis could be shortened by supplementing the sterile filtered proteose peptone medium with high concentrations of nucleotides and glucose [18].Using the conditional mutant NP1, a strain that does not form food vacuoles at the restrictive temperature (37˚C), Rasmussen and Orias showed that at 37˚C, 'mouthless' NP1 could multiply quickly (every 3.5 hours) in a two percent proteose peptone medium supplemented with high concentrations of vitamins and heavy metal salts [17].Their finding further indicates that nucleobases and their derivatives, which are essential for Tetrahymena growth, could enter Tetrahymena cells through the peripheral membrane system.Freeman and Moner fed T. pyriformis GL-7 with [ 3 H]uridine in a short period of time before a new food vacuole could be formed and quantified the amount of radioactive uridine present in different forms in the cell [16].They concluded that uridine could enter through the cell surface and was immediately phosphorylated.
In this study, we tested the growth inhibition of six pyrimidine analogs (5-fluorouracil, floxuridine, 5'-deoxy-5-fluorouridine, 5-fluorouridine, gemcitabine, and cytarabine) on wild-type CU428 and conditional mutant NP1 T. thermophila.Whereas several 5-fluorouracil derivatives have been shown to inhibit Tetrahymena pyriformis growth [6,11], more recently developed pyrimidine analogs, such as gemcitabine, a cytidine analog, and 5'-deoxy-5-fluorouridine, have not been evaluated in Tetrahymena.Furthermore, little is known about how these pyrimidine analogs are transported into and act in the cells.
Our results show that except for cytarabine, the tested pyrimidine analogs are capable of inhibiting T. thermophila growth with different half-maximal inhibitory concentrations (IC50).We also show that phagocytosis is not required for pyrimidine analog uptake.Finally, by supplementing T. thermophila with higher concentrations of exogenous nucleobases and nucleosides in the rescue experiments, some but not all inhibitory effects from the analogs could be reversed.Implications from our study and how our results conform to bioinformatic predictions based on the T. thermophila genome are also discussed [19,20].

Tetrahymena thermophila growth curve measurements
T. thermophila wild-type CU428 (Stock ID: SD00178, MPR1; mp-s, VII) and conditional mutant NP1 strains (Stock ID: SD01422) [21,22] were acquired from the Tetrahymena Stock Center.Cells were maintained and grown at room temperature (air-conditioned at 22˚C) in modified Neff's medium (0.25% proteose peptone, 0.25% yeast extract, 0.5% glucose, and 33.3 μM FeCl 3 ) in Erlenmeyer flasks until the exponential growth phase.The absence of the oral apparatus and phagocytosis of the NP1 strain at the restrictive temperature (37˚C) was confirmed by feeding the cells Congo Red dye and examining the presence of red food vacuoles under light microscope (S1 Fig) .The final cell concentration to start growth curve experiments was approximately 4,000 cells/mL.
Growth inhibition and rescue experiments were conducted in triplicates at room temperature for 48 hours.In the rescue experiments, additional nucleobases (uracil and thymine) or nucleosides (uridine, thymidine, and 5'-methyluridine) were added to T. thermophila culture alongside the pyrimidine analogs in modified Neff's medium.The final concentration of additional nucleobases and nucleosides was set at 5 mM, which was substantially higher than the concentrations of ribonucleotides present in the modified Neff's medium from the yeast extract (4% of the dry weight, or 0.3 mM, according to [23]).Each inhibitory analog was tested at two different concentrations.The inhibitory concentrations of the analogs in the rescue experiments varied: 5-fluorouracil (0.4 mM), floxuridine (0.4 mM), 5'-deoxy-5-fluorouridine (2.5 mM), 5-fluorouridine (0.5 mM), and gemcitabine (0.04 mM).Each analog was also tested at its IC50 concentration.Analogs that might be rescued by additional nucleobases or nucleosides were further tested in an additional concentration: 5-fluorouracil (0.05 mM), floxuridine (0.08 mM), and 5'-deoxy-5-fluorouridine (1.25 mM).Paromomycin and cycloheximide were tested at a final concentration of 10 μg/mL and 5 μg/mL, respectively.
The pyrimidine analogs were each added to T. thermophila cultures four hours after cells were incubated at 37˚C to determine whether phagocytosis is required for analog entry.Cell densities were measured 24 hours after cell inoculation (S2 Fig) using either a WPA CO8000 Cell Density Meter (Biochrom, Cambridge, UK) or a Multiskan FC microplate reader with a 594 nm filter (Thermofisher, Waltham, MA, USA).

Calculation of IC50 values
Data analysis was performed on GraphPad Prism v9.2.0 (San Diego, CA, USA).Half-maximal inhibitory concentrations (IC50) of the tested pyrimidine analogs for CU428 and NP1 were calculated using a nonlinear regression analysis of log(inhibitor) vs. response -variable slope (four parameters).IC50 values for each analog and their standard errors are reported in Fig 1 .Independent-groups t-tests were performed to examine the statistical significance between IC50 concentrations of the same analog on CU428 and NP1.The minimal data set used for calculations and figure creation can be found in the S1 File.

IC50 values of pyrimidine analogs inhibiting CU428 and NP1 growth
Of the six tested pyrimidine analogs, cytarabine (up to 0.5 mM) was the only compound that did not inhibit T. thermophila growth (Fig 1).This is consistent with results reported in T. pyriformis, where no growth inhibition was observed with 0.22 mM of cytarabine (arabinosylcytosine) [6].The other five analogs inhibited CU428 and NP1 cell growth with IC50 values ranging from the low single digits to hundreds of micromolar, with the lowest being 1.61 μM (gemcitabine) and the highest being 654.20 μM (5'-deoxy-5-fluorouridine, Fig 1).Independent-groups t-tests comparing each analog's IC50 values for CU428 and NP1 at room temperature found no significant differences for any of the five analogs (p > 0.3, S1 Table ).
Our findings are consistent with results obtained using T. pyriformis grown in proteosepeptone medium [6], demonstrating that T. thermophila growth can be inhibited by 5-fluorouracil, 5-fluorouridine, and floxuridine (5-fluorodeoxyuridine).In addition, we found that both gemcitabine and 5'-deoxy-5-fluorouridine are effective in inhibiting the growth of T. thermophila.Gemcitabine was recently shown to inhibit the growth of the marine ciliate Euplotes vannus [24], but has not been tested using other ciliate species.

The uptake of pyrimidine bases and nucleosides in T. thermophila does not require phagocytosis
To test whether the uptake of pyrimidine analogs depends on phagocytosis, we cultured both CU428 and NP1 cells at 37˚C, a temperature where the conditional mutant NP1 does not form the oral apparatus required for phagocytosis [21].The absence of phagosomes was confirmed by using a Congo Red stain before the addition of pyrimidine analogs (S1 Fig) .All five analogs inhibited cell growth of both the wild-type CU428 and the 'mouthless' NP1 cells at 37˚C (Fig 1 ), which strongly suggests that the uptake of pyrimidine analogs, including nucleobases and nucleosides in T. thermophila, does not require an oral apparatus and phagocytosis.
Specifically how nucleobases and nucleosides are transported into Tetrahymena remains unknown.Several early studies in T. pyriformis suggested that the transport of these compounds might involve a nucleoside transporter system [16,25,26].Transporter proteins in this system have the ability to move one or more types of nucleotides across membranes, with functional redundancies [16,26].Indeed, results derived from recent bioinformatic analyses suggest that there are more than 10 equilibrative nucleoside transporter genes present in the T. thermophila genome [27,28], indirectly confirming those early observations.However, the functions and locations of these putative transporters, as well as the mode of transport, require further investigation.
No significant differences were found between each analog's IC50 values for CU428 and NP1 at 37˚C (all p > 0.2).The IC50 values of 5-fluorouracil, 5-fluorouridine, and floxuridine on CU428 and NP1 did not change significantly between room temperature and 37˚C (all p > 0.1).In contrast, IC50 values calculated from growth at 37˚C of 5'-deoxy-5-fluorouridine decreased significantly (p = .001)while that of gemcitabine increased significantly (p = .011),when comparing to those values measured at room temperature (Fig 1).We do not have a simple explanation for the changes (or lack thereof) in IC50 values, as the exact inhibition mechanisms for each of the analogs in T. thermophila remain unclear.An analog may act on one or more enzymes, and may have varying binding affinities to enzymes at different temperatures.

Growth inhibitory effects may be reversed by adding additional nucleobases or nucleosides
To further investigate how the five pyrimidine analogs might affect pyrimidine metabolism in T. thermophila, we added additional pyrimidine bases and nucleosides to determine whether they could compete against the analogs and reverse the growth inhibition.Two pyrimidine bases (uracil and thymine) and three nucleosides (uridine, thymidine, and 5-methyluridine) were used in the rescue experiments; the nucleobases and nucleosides were added alongside the pyrimidine analogs to T. thermophila culture.We expected that higher concentrations of uracil and uridine might outcompete 5-fluorouracil and its derivatives, but not gemcitabine, which is a cytidine analog.Furthermore, if thymidylate synthase, which synthesizes thymidine for DNA replication by converting dUMP to dTMP, is the primary inhibitory site for 5-fluorouracil and its derivatives as shown in human cancer cells [29,30], supplying T. thermophila with additional thymine or thymidine should mitigate the inhibitory effect (Fig 2).We did not expect 5-methyluridine to reverse the inhibitory effects since it is inert in supporting the growth of T. pyriformis [11][12][13].In addition, two antibiotics, paromomycin and cycloheximide, that inhibit the growth of Tetrahymena by blocking protein synthesis [31,32], are not expected to be rescued from any of the supplementary pyrimidine bases or nucleosides.
In the rescue experiments, the concentration of the five rescuing pyrimidine bases and nucleosides was fixed at 5 mM.This concentration was substantially higher than the two concentrations of each pyrimidine analog tested: an inhibitory concentration and the IC50 (see Materials and Methods).For 5-fluorouracil, floxuridine, and 5'-deoxy-5-fluorouridine, whose inhibitory effects were found to be reversible in the rescue experiments, we tested an intermediate concentration between the inhibitory concentration and the IC50.
As predicted, 5-methyluridine was not able to reverse the inhibitory effects from any of the five analogs (Figs 3-6) and none of the five pyrimidine bases and nucleosides we tested could reverse the inhibitory effect of gemcitabine and the two antibiotics (Fig 6A , 6B, 6E and 6F).Additionally, growth inhibition from 5-fluorouridine could not be reversed by any of the five pyrimidine bases and nucleosides (Fig 6C and 6D).In the absence of the pyrimidine analogs, none of the additional pyrimidine bases or nucleosides affected T. thermophila growth (Figs 3D, 4D and 5D).
We found that uracil but not uridine, and thymine but not thymidine, could reverse the inhibitory effects from 5-fluorouracil (Fig 3).Uracil was capable of reversing the inhibitory effect at all three 5-fluorouracil concentrations (Fig 3A -3C).The rescue effect of thymine was most profound at the intermediate concentration of 5-fluorouracil (0.05 mM, Fig 3B ), but not at a higher, inhibitory concentration (0.4 mM, Fig 3A).
Growth inhibition from floxuridine could only be reversed by thymidine at the intermediate concentration and IC50 (Fig 4), and those from 5'-deoxy-5-fluorouridine could only be reversed by uracil at the two lower concentrations (Fig 5).
While early studies suggested that uracil and uridine were equally effective as the sole pyrimidine source supporting Tetrahymena growth [11], our rescue experiments showed that only uracil was capable of reversing the growth blockages in T. thermophila caused by 5-fluorouracil and its prodrug 5'-deoxy-5-fluorouridine (Figs 3 and 5).This finding suggests that uracil and uridine are not as interchangeable in Tetrahymena as once thought.One clue for this non-interchangeability may be found in the Tetrahymena genome [20] as the key enzyme responsible for interconverting uridine and uracil is absent, according to the KEGG pathway (S3 Fig) [19].However, results derived from several early experiments strongly suggest the presence of uridine phosphorylase activity in T. pyriformis [33][34][35].Whether these discrepancies indicate the presence of a non-canonical uridine phosphorylase in Tetrahymena warrants further investigation.Our results suggest that uridine and uracil are differentially metabolized in T. thermophila: uridine may be preferentially used in RNA synthesis and uracil for DNA synthesis when both are available.Because 5-fluorouracil and 5'-deoxy-5-fluorouridine  [36]), uracil may be more effective in reversing their inhibitory effects.
Interestingly, in addition to uracil and uridine, thymine and thymidine also appear to be differentially metabolized in Tetrahymena.This was first indicated in an early experiment where thymidine, but not thymine, was shown to reinitiate growth of T. pyriformis grown in defined medium lacking folic acid [11].We show that thymine, but not thymidine, reversed the growth blockage from 5'-deoxy-5-fluorouridine in T. thermophila grown in modified Neff's medium (Fig 5).Thymidine, but not thymine, could similarly reverse the growth blockage from floxuridine (Fig 4).Altogether, these observations strongly suggest inefficient interconversions between thymine and thymidine, despite the biochemical and bioinformatic detection of thymidine phosphorylase activity in Tetrahymena (S3 Fig, EC 2.4.2.4) [37].Indeed, it has been shown that thymidine is a much more effective precursor than thymine to be incorporated into DNA in Tetrahymena among other eukaryotic cells [38,39].Finally, the fact that thymine, but not thymidine, could reverse the growth blockage from 5'-deoxy-5-fluorouridine indicates that this nucleobase might be able to be converted to TMP without going through a thymidine intermediate.However, other mechanisms (e.g., competitive or allosteric regulation) to interpret our results remain plausible.
It is noteworthy that our results show that thymidine, but not uridine, rescued the growth of T. thermophila from floxuridine blockage in modified Neff's medium (Fig 4).This would contradict the results from T. pyriformis grown in a defined medium that uridine, but not thymidine, reversed the growth blockage from the same analog [11].It is not clear what may have caused the discrepancies.The concentrations of floxuridine used in both studies were similar while our experiments used a much higher concentration of rescuing pyrimidines (5 mM) compared to approximately 41 μM (10 mg/mL) in Wykes and Prescott's study [11].In addition, the Tetrahymena species and growth media used in the two studies were different.More experiments are clearly needed to further elucidate the blockage mechanisms of floxuridine and other pyrimidine analogs and the mechanism of nucleotide transport and metabolism in different Tetrahymena species.

Conclusions
In this study, we demonstrated that 5-fluorouracil, floxuridine, and 5-fluorouridine inhibited the growth of T. thermophila grown in modified Neff medium (compared to T. pyriformis in a defined medium in previous studies).Furthermore, we showed that both gemcitabine and 5'deoxy-5-fluorouridine inhibit T. thermophila growth, while cytarabine does not.We present new evidence suggesting that these pyrimidine analogs may enter T. thermophila through their peripheral membrane systems.Results obtained from the rescue experiments suggest differential metabolism of uracil and uridine, and thymine and thymidine in T. thermophila.

Fig 2 .Fig 3 .
Fig 2. Schematic drawing of uracil and uridine pathways in T. thermophila.The question mark indicates an absence of a uridine phosphorylase homolog interconverting uridine and uracil through bioinformatic searches, but such enzyme activity was strongly suggested by biochemical experiments (see main text).https://doi.org/10.1371/journal.pone.0284309.g002